Resistance to downy  mildew of onion caused by the fungus peronospora destructor

ABSTRACT

The present invention relates to plants of the  Allium cepa  species, which are resistant to the downy mildew of onion caused by the fungus  Peronospora destructor , due to a Pd resistance locus, wherein any fragment of a chromosome, comprising the Pd resistance locus, can be present homozygously in progeny without causing lethality. The present invention also encompasses plants of the  Allium cepa  species which are resistant to downy mildew of onion caused by the fungus  Personospora destructor  (Pd) due to Pd resistance locus present homozygously in the genome of the plants. The present invention also provides processes for obtaining plants resistant to downy mildew of onion, which are suitable for obtaining cultivated onions and shallots.

The present invention relates to plants of the genus Allium which areresistant to the downy mildew of onion caused by the fungus Peronosporadestructor (Pd) (Berk.) Cas., specifically plants of the Allium cepa orAllium fistulosum species. According to the invention, the resistance isprovided by a resistance locus, which can be present homozygously orheterozygously in the genome of the plant, and which is sufficient toprovide resistance to plants bearing this locus. The present inventionalso provides processes for obtaining said plants resistant to downymildew of onion, said processes being suitable for obtaining cultivatedonions and shallots.

BACKGROUND OF THE INVENTION

Downy mildew caused by the fungus Peronospora destructor (Pd) ispractically world-wide in distribution. The pathogen attacks variouskinds of onions, but is especially destructive to the common onionAllium cepa. The damage caused by the fungus is described in Mukerji(ref 5). Spontaneous epidemics may occur in the field, if conditions arefavourable for the disease. The symptoms are yellowing of the leaves andgrey sporulation.

From an economical point of view, downy mildew is one of the majorfungal diseases threatening the cultivation of onions and shallots(Allium cepa L.), almost in all onion-growing regions in the world.

It is assumed that no complete natural resistance to downy mildew ispresent in Allium cepa and Allium fistulosum. However, a completeresistance to downy mildew was found in the wild Allium roylei Stearn.Inspired by a certain morphological resemblance between A. roylei and A.cepa it has therefore been proposed to use A. roylei as an introgressionpartner for A. cepa. In the publication by van der Meer and de Vries(ref 1) preliminary results are reported concerning such a hybridbetween A. roylei and A. cepa, as well as results concerning thebackcross of the interspecific hybrid to A. cepa. It has been observedthat said interspecific hybrid is male and female fertile (Kofoet et al,ref 2). It has also been reported that a downy mildew resistance can bedominantly inherited in the BC1 (first backcross) progeny of this hybridto onion.

The locus in the cepa genome, where the introgressed sequencesresponsible for the resistance are found, has been termed “Pd resistancelocus”. By extension, “Pd resistance locus” also designates thesequences themselves.

However, segregation for downy mildew resistance among BC1 and F2progenies from the F1 between A. roylei and A. cepa is observed (deVries et al, ref 3) and no breeding onion varieties resistant to downymildew have ever been obtained, more than 10 years after the firstresistant hybrid was obtained.

There is thus great interest from an agronomical and economical point ofview for plants of Allium cepa and Allium fistulosum species, which areresistant to downy mildew and which are still 100% resistant afterself-pollination i.e. there is no segregation of the resistancecharacteristic. These plants are particularly valuable as they mayindeed be crossed with a susceptible line, giving hybrids which are alsoresistant to downy mildew of onion.

The invention lies first in the observation that all available resistantmaterial of the Allium cepa and Allium fistulosum species was in factheterozygous for the Pd resistance sequences and that no homozygousresistant plants have been obtained or disclosed in a reproduciblemanner up to now.

The present inventors elucidated the reason for which no homozygouslyresistant plant could be obtained and then succeeded in obtaining suchAllium plants, which gives rise to progeny after self-pollination whichis also 100% resistant (i.e. a homozygously resistant plant).

Several hypotheses could have explained the striking finding that nohomozygous plants were available: translocation, recombination, hybridpreference selection, gene silencing and pleiotropic effects, linkagedrag, etcetera.

The present inventors have determined that the introgression fragment ofAllium roylei which is present in the cepa hybrid and confers resistanceto downy mildew comprises also a sequence designated “lethal factor”,whose presence on both homologues of chromosome in cepa is lethal to theplant. For a resistant cepa plant to exist and grow, the introgressionfragment, conferring resistance and containing the lethal factor, isthus necessarily present on a single chromosome homologue, explainingthe absence of obtained homozygously resistant plants by thepredecessors. This sequence responsible for lethality is present in thevicinity of the Pd resistance locus.

Having identified that the sequence conferring resistance is linked to alethal sequence, the present inventors have succeeded, for the firsttime, in physically separating the sequence conferring resistance fromthe sequence responsible for lethality when present on both chromosomes.

This step of separation, which can be brought about by a recombinationevent, is the key point of the present invention. Indeed it could not beforeseen that the sequences conferring resistance could be separatedfrom the lethal factor. First of all, it was not known whether saidfactor was a sequence which in itself was lethal or whether lethalityarose as a result of the replacement, by sequences present on anintrogression fragment, of endogenous essential sequences. Indeed, it ispossible that the lethality is in fact a knock out of essential gene(s)on the corresponding cepa-fragment, which are not compensated by thefragment introduced from A. roylei. Therefore, different scenarios werepossible to explain lethality:

-   -   the resistance sequences might have constituted in fact the        lethal factor, because they replace endogenous critical        sequences. They could thus never be present on both chromosomes        without causing lethality.    -   the resistance sequences and the lethal sequences might have        been extremely near to each other on the chromosome, or even        overlapping. In this situation, the probability of obtaining a        recombination event separating both is negligible.    -   the sequences constituting the lethal factor might have been        necessary for the functioning of the resistance. In this        condition, separating both would lead to the loss of the        capacity for resistance.

In this context, the present inventors have unexpectedly succeeded inseparating the sequences conferring the resistance phenotype from thesequence whose presence on both homologues is lethal.

The inventors have also found that the already released resistant A.cepa possess both the Pd resistance locus (which is dominant) and thelethal factor (which is recessively inherited) on the same introgressionfragment. As the Pd resistance locus is dominant, the phenotype of theplant is “resistant” and the recessive lethal factor is not observed.However, progeny of such plants when crossed with susceptible plantswill segregate and thus cannot be used commercially. Indeed, commercialonions and shallots are generally varieties, thus plant material,whether lines or hybrids, with a phenotype which has to be uniform;segregating traits of interest cannot be envisaged for commercialplants.

The present invention thus provides plants of the Allium cepa and Alliumfistulosum species which are resistant to downy mildew of onion causedby Peronospora destructor (Pd) due to the presence in their genome of aPd resistance locus, and said plants are homozygously resistant plants,i.e. plants which give a progeny after selling which is also 100%resistant.

The present invention also provides resistant plants which can beobtained by crossing the above-mentioned resistant plants of the Alliumcepa or Allium fistulosum species with a plant also of the Allium cepaor Allium fistulosum species which is susceptible to downy mildew.

The present invention also provides processes for obtaining said plants.

Definitions

In the context of the present application, the following terms aredefined in the following manner:

Introgression: natural introduction of genes of one species into anotherthrough the process of interspecific hybridization followed bysuccessive backcrosses to the recurrent parents. Each species maythereby become more variable and show certain characters of the otherspecies.

Pd resistance locus: Place in the chromosome occupied by the sequencesresponsible for resistance to Peronospora destructor, said sequencesbeing sufficient to confer resistance to a plant. A Pd resistance locusmay contain one gene or several genes, possibly separated by unrelatedsequences or genes. By extension, in the context of the presentinvention, the sequences themselves are also called Pd resistance locus.

A possible Pd resistance locus is the introgression fragment of A.roylei in the genome of a plant of the line 3591-1 NCIMB accessionnumber 41249.

Susceptible/susceptibility: according to the International SeedFederation, Section Vegetables, Position Paper of May 2004, in“Definition of the Terms Describing the Reaction of Plants to Pests orPathogens for the Vegetable Seed Industry”, susceptibility is theinability of a plant variety to restrict the growth and development of aspecified pest or pathogen. It must however be noted that the term“sensitive” has also been widely used in the past decades for describingthe same property.

By contrast, a plant which is able, to a certain extent and whencompared to susceptible plant varieties under similar environmentalconditions and pest or pathogen pressure, to restrict the growth anddevelopment of a specified pest or pathogen is qualified as resistant tosaid specified pest or pathogen. Resistant plants include plants whichare tolerant, i.e. remain infected but survive, and plants which aretotally resistant. Although being resistant to a pest or pathogen, aresistant plant attacked by said pest or pathogen may display symptomscharacteristic of the infections, like reduced growth, earlier death,loss of leaves . . .

Inbred line: nearly homozygous line (for all characters) produced bycontinued inbreeding and selection.

Homozygously resistant plant to a pathogen: plant which gives a progenyafter selfing (self-pollination) which is also 100% resistant to saidpathogen. Where the resistance is due to the presence of a DNA sequencein a chromosome of the plant, said sequence is present on all thehomologues of the chromosome.

Lethal factor: factor, for example a genetic factor, which hinders thesurvival of a plant exposed to this factor. The presence of a lethalfactor may prevent the plant from existing ab initio or may cause itsdeath at a later stage.

Allium cepa: Species of the Allium genus, comprising bulbous plantshaving hollow leaves cultivated worldwide for its rounded edible bulb.Cultivar onions and shallots are Allium cepa species.

Recombination: Crossing-over occurring during meiosis.

The present invention provides a first type of plant, that is a plant ofthe Allium cepa or Allium fistulosum species which is resistant to downymildew of onion caused by the fungus Peronospora destructor (Pd).According to the present invention, the resistance to downy mildew isdue to a Pd resistance locus in the genome of the plant, characterizedin that the resistance locus is present homozygously in the genome, thatis the sequences responsible for the resistance are present in twocopies in the genome of a plant according to the invention, i.e. presenton both chromosomal homologues.

Indeed, the present inventors have succeeded in separating the sequencesresponsible for the resistance to downy mildew from the linked lethalsequences, which were identified by the inventors as being lethal ifpresent on both chromosome homologues. By separating these two types ofsequences, the inventors have thus been able to obtain a planthomozygous for the sequences of interest (Pd resistance locus), withoutbeing homozygous for the deleterious sequences (lethal factor). Theinvention concerns these viable plants homozygous for the sequencesconferring resistance. These plants are necessarily not homozygous forthe lethal sequences, which may however be present, but at only a singlecopy.

As these plants according to the invention are homozygous for thesequences of interest conferring resistance, any progeny of a plant ofthe invention will lead to a plant also resistant to downy mildew,without segregation of this feature. Indeed, any plant in the progenywill have in its genome the Pd resistance locus, which is known to bedominant over the susceptible phenotype.

A plant is considered to be resistant if it is able to restrict thegrowth and development of Peronospora destructor, by hindering theinfection or combating the growth of the fungus after infection. Bycontrast, a plant which is unable to restrict the growth and developmentof Peronospora destructor is said to be susceptible to Peronosporadestructor.

Said resistance according to the invention comprises resistance tonatural infection by Peronospora destructor and also resistance toartificial inoculation by Peronospora destructor. Artificial tests canbe carried out in greenhouses or climate rooms under more controlledenvironments. Both young plants and sprouting bulbs can be tested.

Protocols for testing whether a plant is resistant or susceptible toPeronospora destructor are reported in the article by de Vries et al(ref 3).

Resistance may also be tested genetically with molecular markers, byassaying the presence of the Pd resistance locus in the genome of aplant. The techniques which can be used are well known to the skilledman in the domain and are generally based on genic amplification.

Techniques which can be used for molecular typing include restrictionfragment length polymorphism (RFLP), multilocus enzyme electrophoresisand random amplified polymorphic DNA (RAPD). Another technique which isparticularly suitable for testing the presence of the Pd resistancelocus in the genome of a plant, is the AFLP™ (Amplified Fragment LengthPolymorphism); such a technique is described for example in Vos et al(ref 4). This technique consists in a first step of digestion of thegenomic DNA with suitable restriction enzymes, simultaneously withligation with specifically designed oligonucleotide adapters. Theadapters have sequences corresponding to the restriction site, linked toan additional defined sequence. In a second step, PCR amplification ofthe obtained tagged restriction fragments is then carried out withprimers, whose sequence comprises a sequence complementary to theadapters and 1, 2 or 3 additional nucleotides allowing discriminationbetween the tagged restriction fragments. This PCR amplification allowsdetection of restriction fragment length polymorphisms.

In example 4 below are given four specific pairs of primers which giveamplified fragments of defined length, only when the Pd resistance locusis present. If a plant generates one or more of the expected amplifiedfragments, it can be deduced that this plant possesses the whole Pdresistance locus and is thus resistant to downy mildew. The use ofmolecular markers is particularly well suited in the context of thepresent invention.

Preferably, resistant plants according to the invention are resistant toinfection by Peronospora destructor.

Plants according to this first type according to the invention are forexample plants from the line 3591-1 which has been deposited at theNCIMB under accession number 41249. The process by which the inventorshave obtained this plant is reported in example 1.

The present invention also concerns a second type of plants, which areplants possessing said Pd resistance locus heterozygously, i.e. on onlyone of the chromosome homologue. According to the invention, said plantscan be obtained by crossing the homozygous plant mentioned above with asecond plant of the Allium cepa or Allium fistulosum species, the secondplant being susceptible to downy mildew of onion.

Depending on the species of the plants which are crossed, the inventionthus encompasses hybrids between Allium cepa and Allium fistulosum, aswell as plants of the Allium fistulosum species and plants of the Alliumcepa species.

Due to the fact that the Pd resistance locus is present heterozygouslyin the second type of plants of the invention, the resistance traitsegregates when further to self-pollinations or crossing them with otherplants.

A plant according to this embodiment of the invention can be obtained bycrossing a plant from the line 3591-1 which has been deposited at theNCIMB under accession number 41249, with a downy mildew-susceptibleAllium cepa parental line. This parental line is preferably acytoplasmic male sterile Allium cepa inbred line.

The second type of plant according to the invention can of course beobtained by other processes. For example, they can also be obtained byself-pollinating plants already of this type, although, as explainedabove, progeny of such a self-pollination is not 100% resistant to downymildew.

According to the present invention, the Pd resistance locus ispreferably present on chromosome 3, homozygously or heterozygously, inthe genome of plants according to the first or second type of thepresent invention. Plants of the Allium cepa and Allium fistulosumspecies have indeed 2n=16 chromosomes which are considered to behomoeologous chromosomes (see in particular reference 8).

Plants of the invention also includes plants of the Allium cepa orAllium fistulosum species, which are resistant to downy mildew of onioncaused by the fungus Peronospora destructor (Pd) due to a Pd resistancelocus, wherein any fragment of the chromosome comprising the Pdresistance locus, can be present homozygously in progeny without causinglethality.

As mentioned above, in the proximity of the Pd resistance sequences orlocus, sequences causing lethality may generally be found. The portionof the chromosome comprising the Pd resistance locus and said lethalsequences may generally not be present on both chromosome homologues,because it prevents the plant from existing. By disjoining the Pdresistance locus from the lethal sequences, the inventors have been ableto obtain plants which contain the Pd resistance locus and wherein anyportion of the chromosome comprising the Pd resistance locus is or maybe present on both chromosome homologues, because the lethal sequencesare not present.

Plants of the Allium fistulosum species according to the invention maybe obtained by crossing a first plant of the line 3591-1 (deposited atthe NCIMB under accession number 41249) with a plant of the Alliumroylei species and then backcrossing the obtained hybrid with plants ofthe Allium fistulosum species, one or several times, that is usingAllium roylei as a bridge species.

The invention also concerns a plant of the Allium cepa or Alliumfistulosum species, which is resistant to downy mildew of onion causedby the fungus Peronospora destructor (Pd), wherein at least one of thehomologues of chromosome 3 comprises a Pd resistance locus and anyfragment of said chromosome, comprising the Pd resistance locus, can bepresent homozygously in progeny without causing lethality.

It must be noted that a plant being homozygous for the Pd resistancelocus and heterozygous for the lethal sequences, i.e. the lethalsequences are present on only one homologue of chromosome 3, is alsopart of the present invention. Indeed, any fragment of at least one ofthe homologue of chromosome 3, the homologue comprising the Pdresistance locus without the lethal sequences, can be presenthomozygously in progeny without causing lethality.

Such a plant is for example plants 3591-3, 3591-4, 3591-5, 3591-6 or3591-8 exemplified in example 3 of the experimental section of thepresent application.

By self-pollinating a resistant plant, it can be deduced by analysis ofthe progeny whether the plant corresponds to the definition given above.Example 5 gives details about the analysis to be carried out for such adetermination. The main criterion to be evaluated is the percentage ofprogeny which is resistant. Any resistant plant is a plant according tothe invention if at least 75% (+/−5% due to variability of the measures)of the progeny is also resistant. FIG. 1 exemplifies the differentsituations at the genomic level, leading to this percentage.

It must be noted that a plant having a Pd resistance linked to lethalsequences on only one chromosome homologue is not part of the presentinvention. Such a plant, after self-pollination, will give a progenywhich is, according to the genetic law, 66.7% resistant, i.e. less than75%. Such a plant can thus be discriminated from plants according to thepresent invention.

Such a plant is for example plant of line 2348 exemplified in example 2of the experimental section of the present application.

Preferably, the Pd resistance locus is present homozygously in a plantaccording to the invention.

According to a preferred embodiment of the present invention, the Pdresistance locus, which confers resistance to infection by Peronosporadestructor, is present on an introgression fragment; it originates froma plant which is naturally resistant to downy mildew of onion andpreferably from a wild Allium genitor resistant, most preferably fromAllium roylei. The presence of an introgression fragment in the genomeof a plant of Allium cepa or Allium fistulosum species can be obtainedby crossing said plants with an introgression partner, thus obtaining ahybrid. Said hybrid is preferably crossed back with plant of Allium cepaor Allium fistulosum species in order to minimize the introgressionfragments, and then selected on the capacity to show resistance to downymildew.

Indeed, the introgression fragments in a plant of Allium cepa or Alliumfistulosum species according to the invention are preferably limited inorder to have plants which share all the characteristics of interest ofthe parental plant but the resistance trait.

Preferably, introgression fragments in the genome of a plant accordingto the present invention are only present in one or both homologues ofchromosome 3 and preferably in the long arm of the chromosome 3. It ispreferred that the introgression fragment comprising the Pd resistancelocus is less than 50% of the length of the long arm of chromosome 3,preferably less than 44%, more preferably less than 35, 30, 25 or 20% ofthe length of the long arm of chromosome 3. As mentioned above, theintrogression fragment preferably originates from Allium roylei genome.

Plants according to this type are plants from the line 3591-1 which hasbeen deposited at the NCIMB under accession number 41249.

It should be noted that one, two or more introgression fragments can bepresent, for example in the long arm of chromosome 3, however eachfragment being distinct and disjoined from the others. There ispreferably only one introgression fragment.

According to the most favourable situation, the introgression fragmentpresent In a plant according to the invention is only the sequencesstrictly necessary to the resistance to downy mildew.

Plants according to the invention can be used to transfer the Pdresistance into other agronomical valuable plants, provided the plantscan be crossed together. This may be done by obtaining a hybrid and thenbackcrossing the hybrid with the second plants, followed byself-pollinations of the obtained plants; at each step, resistantprogeny is selected.

Preferably, plants according to the invention are stable for thecharacteristic of interest, i.e. resistance to downy mildew of onion,however not necessarily for other traits.

A plant according to the present invention may be obtained from aninitial interspecific cross between a wild Allium genitor resistant todowny mildew of onion and Allium cepa. According to this situation, theintrogression fragment originates from the wild Allium genitor.

In order to obtain a plant according to the invention, that is of theAllium cepa or Allium fistulosum species, it must be necessary to selecthybrid plants resulting from the cross which share the largestphenotypic similarity with the parent of the Allium cepa or Alliumfistulosum species however being resistant to downy mildew. Preferably,a plant according to the invention can be obtained after the initialinterspecific cross mentioned above followed by several backcrosses withplant of the parental species and optionally one or severalself-pollinations.

The steps of backcrosses and self-pollinations allow to reduce theproportion of introgression fragments in the resulting plant. The numberof backcrosses which is recommended is at least 2, preferably 3, 4, 5 or6, 7, 8 to 10 successive backcrosses may also be envisaged. The numberof self-pollinations which are to be performed is between 1 and 8,preferably 2, 3, 4 or 5.

According to a preferred embodiment of the present invention, theinitial interspecific cross is between wild Allium genitor resistant todowny mildew of onion and Allium cepa and the mentioned backcrosses arecarried out with plants of the Allium cepa species.

The wild Allium genitor is any plant of Allium genus which is resistantto downy mildew of onion caused by Peronospora destructor, preferably anaturally resistant one, and a preferred Allium genitor is a plant ofthe Allium roylei species.

As mentioned above, the presence of the Pd resistance locus can betested according to well known protocols for detecting the resistance toinfections by Peronospora destructor. These protocols however aretime-consuming. The presence of the Pd resistance locus may thus also betested by other genetic techniques, based on genic amplification, whichare well known to the skilled man in the domain.

Techniques which can be used for molecular typing include restrictionfragment length polymorphism (RFLP), multilocus enzyme electrophoresisand random amplified polymorphic DNA (RAPD). Another technique which isparticularly suitable for testing the presence of the Pd resistancelocus in the genome of a plant, is the AFLP™ (Amplified Fragment LengthPolymorphism); such a technique is described for example in reference 4.This technique is particularly suitable for the present invention.

According to the present invention, this technique called AFLP™ is usedfor assaying the presence of the Pd resistance locus in the genome of aplant of Allium genus. The restriction of the genomic DNA of the plantis preferably performed with the restriction enzymes PstI and MseI; andthe ligation is performed with the following adapters:

alpha 5′-CTCGTAGACTGCGTACATGCA CATCTGACGCATGT-5′, and beta5′-GACGATGAGTCCTGAG TACTCAGGACTCAT-5′.

The PCR amplification is then carried out on the restriction fragmentsligated to the adapters with the following primers:

(A) 5′-GACTGCGTACATGCAGAAC and 5′-GATGAGTCCTGAGTAACTT.

According to this procedure, the presence of a Pd resistance locus inthe tested plant can be deduced from the presence of an amplifiedfragment of 61 nucleotides. Indeed, if such a fragment results from thepreceding steps, it is indicative of the presence of a Pd resistancelocus according to the invention, as can be deduced from the resultsreported in the example 4.

Alternatively, the method can be carried out according to the followingalternative protocol:

-   -   the restriction of the genomic DNA of the plant is performed        with the restriction enzymes PstI and MseI; and    -   the ligation is performed with the following adapters:

alpha 5′-CTCGTAGACTGCGTACATGCA CATCTGACGCATGT-5′, and beta5′-GACGATGAGTCCTGAG TACTCAGGACTCAT-5′;

-   -   the PCR amplification is amplification is then carried out on        the restriction fragments ligated to the adapters with the        following primers:

(B) 5′-GACTGCGTACATGCAGAAG and 5′-GATGAGTCCTGAGTAAAAC.

When such a protocol is carried out, a specific fragment of 151nucleotides is representative of the presence of a Pd resistance locusin the genome of the tested plant, as can be deduced from the resultsreported In the example 4.

According to a third alternative, the method can be carried outaccording to the following protocol:

-   -   the restriction of the genomic DNA of the plant is performed        with the restriction enzymes PstI and MseI; and    -   the ligation is performed with the following adapters:

alpha 5′-CTCGTAGACTGCGTACATGCA CATCTGACGCATGT-5′, and beta5′-GACGATGAGTCCTGAG TACTCAGGACTCAT-5′;

-   -   the PCR amplification is amplification is then carried out on        the restriction fragments ligated to the adapters with the        following primers:

(C) 5′-GACTGCGTACATGCAGACA and 5′-GATGAGTCCTGAGTAACCA.

When such a protocol is carried out, a specific fragment of 330nucleotides is indicative of the presence of a Pd resistance locus inthe genome of the tested plant, as can be deduced from the resultsreported in the example 4.

Example 4 of the present invention describes the use of such a method.

The present invention is also directed to a plant of the Allium cepaspecies, homozygously resistant to the downy mildew of onion, i.e. theprogeny of said plant after self-pollination is 100% resistant to downymildew, which can be obtained by carrying out the following process:

-   -   a) Interspecific cross between Allium roylei and Allium cepa;    -   b) Selection of an interspecific hybrid, resistant to downy        mildew of onion caused by the fungus Peronospora destructor        (Pd);    -   c) Backcross of said hybrid with plants of Allium cepa;    -   d) Selection of plants resistant to downy mildew of onion;    -   e) Self-pollination of the thus obtained plants;    -   f) Selection of plants homozygously resistant to downy mildew of        onion.

The step a) has been proven to give viable interspecific hybrids whichare male and female fertile. The interspecific cross can be made betweenAllium roylei as the male and Allium cepa as the female parent or on thecontrary with Allium roylei as the female and Allium cepa as the maleparent.

The step b) is a step of selection of plants resistant to downy mildewof onion caused by the fungus Peronospora destructor. As mentioned inthe preceding paragraphs, the resistance may be tested by natural orartificial inoculations with known protocols described in the literatureor in the experimental section of the present application. The selectionmay also be achieved by taking advantage of the AFLP™ method describedabove. Useful restriction enzymes, adapters and PCR primers aredescribed above, along with the length of the expected amplifiedfragment indicative of the presence of Pd resistance locus.

According to the present invention, the plants selected at the step b)not only are resistant to downy mildew of onion, but they share alsoadvantageously as many as possible morphological characteristics withthe Allium cepa parent.

Step c) is concerned with backcross of the interspecific hybrid selectedat step b) for its resistance to downy mildew of onion with plants ofthe Allium cepa species. These Allium cepa plants used for the backcrossstep may be from the same subspecies, variety or form than the plantused in step a) as parent for the interspecific hybrid, but notnecessarily.

After plants resulting from the backcross are obtained, plants areselected which are resistant to downy mildew of onion. As mentioned forstep b), this selection may be achieved by testing the capacity toresist an infection by Peronospora destructor, or by achieving moleculartyping, for example by the AFLP™ technique.

In a preferred embodiment of the present invention, this step c)followed by step d) are advantageously repeated at least once, that isthere are at least two successive backcrosses. The number of backcrossesmay vary between 2 to 10, preferably 3, 4, 5 or 6.

The step e) is a step of self-pollination or selfing, which is wellknown by one skilled in the art.

This step of self-pollination is followed by selection of plantsresistant to downy mildew of onion, which share all the traitscharacteristic of a plant of the Allium cepa species.

Preferably, these steps of self-pollination and selection are repeatedat least once, i.e. at lest two series of self-pollination and selectionare achieved. These steps are preferably carried out 2, 3, 4, 5 or 8times, i.e. they are repeated 1, 2, 3, 4 or 7 times.

The last step of the process concerns the selection of a plant which ishomozygously resistant to downy mildew of onion caused by Peronosporadestructor. This homozygosity may be tested by achieving step e) andchecking that 100% of the progeny is resistant to downy mildew.

According to the present invention, this last step of selection may beachieved preferably by using a molecular typing technique, for examplethe AFLP™ technique as described in the preceding sections andpreferably with the restriction enzymes, adapters and primers recitedbefore.

The present invention also concerns a method for the production of aplant of the Allium cepa species which is resistant to downy mildew ofonion caused by the fungus Peronospora destructor (Pd) which comprisesthe following steps:

-   -   a) Obtaining an Allium roylei resistant to downy mildew of        onion;    -   b) Making a interspecific cross between said Allium roylei and        an Allium cepa;    -   c) Selecting an interspecific hybrid resistant to downy mildew        of onion;    -   d) Backcrossing said hybrid with plant of Allium cepa;    -   e) Selecting plants resistant to downy mildew of onion;    -   f) Self-pollinating the resistant plant obtained at step e);    -   g) Selecting plants homozygously resistant to downy mildew of        onion.

According to the present invention molecular markers are used in stepsc), e) and/or g) for selecting plants resistant to downy mildew ofonion.

The different steps of the method are as explained in the precedingparagraphs. The preferred embodiments of the process were also mentionedpreviously, in particular, the repetition of the steps d) and e) and ofthe steps f) and g). The steps of selection may also be achieved asexplained previously. They are preferably achieved using a moleculartyping method making use of molecular markers. A particularly preferredmethod is AFLP™ which is described in the preceding sections. Accordingto the invention, molecular markers may be used in the selection step c)only, or in the step e) only, or in step g) only, or in each of thesesteps, or in two of three.

At each selection step, the selected plants are preferably plantssharing the highest number of traits characteristic for Allium cepa.According to this procedure, the introgression fragments originatingfrom the first Allium roylei genitor are minimized at each step. Thetraits characteristic for Allium cepa are as disclosed in the Guidelinesfor the Conducts of Tests for Distinctness, Uniformity and Stability(reference TG/46/6), in connection with the treaty UPOV.

According to the invention, the method may also comprise an additionalstep of crossing the plant obtained at the end of step g) with a plantof Allium cepa species which is susceptible to downy mildew of onion.The resulting plant is resistant to downy mildew, as the plant obtainedat the end of step g) is homozygously resistant to downy mildew.

As already mentioned, the selection step c), e) and/or g) may be carriedout using the AFLP™ technique. As exemplified in the presentapplication, the restriction enzymes used in this technique arepreferably enzymes PstI and MseI. Suitable pair of adapters for theligation step is:

alpha 5′-CTCGTAGACTGCGTACATGCA CATCTGACGCATGT-5′, and beta5′-GACGATGAGTCCTGAG TACTCAGGACTCAT-5′.

For the PCR amplification step, suitable pairs of primers which can beused are:

(A) 5′-GACTGCGTACATGCAGAAC and 5′-GATGAGTCCTGAGTAACTT; (B)5′-GACTGCGTACATGCAGAAG and 5′-GATGAGTCCTGAGTAAAAC; and (C)5′-GACTGCGTACATGCAGACA and 5′-GATGAGTCCTGAGTAACCA.

Several fragments are amplified and can be detected on an agar gel afterthis PCR amplification. However, the presence of a fragment having 61nucleotides when the pair of primers (A) is used, is indicative of thepresence of a Pd resistance locus in the genome of the tested plant.Alternatively, when the pair of primers (B) is used, the length of theindicative amplified fragment is 151 nucleotides, and 330 when the pairof primers (C) is used.

In a preferred embodiment of the invention, when using the AFLP™technique, additional PCR amplifications may also be carried out, inorder to detect false positives. According to this procedure,amplifications are carried out with at least one pair of primers chosenamongst the following pairs of primers:

(A′) 5′-GACTGCGTACATGCAGAAG and 5′-GATGAGTCCTGAGTAAAAC; (B′)5′-GACTGCGTACATGCAGAAC and 5′-GATGAGTCCTGAGTAACTT; and (C′)5′-GACTGCGTACATGCAGAAA and 5′-GATGAGTCCTGAGTAACAC.

The amplified fragments resulting from these additional amplificationsare used as negative controls. No fragment of the length expected whenusing pair of primers (A), (B) or (C) must be detected when using pairof primers (A′), (B′) or (C′).

The present invention also encompasses plants of the Allium genus whichcan be obtained by the methods as described, in particular by the methodaccording to the preferred embodiments.

The invention also concerns a hybrid Allium cepa plant obtainable bycrossing a downy mildew-susceptible Allium cepa parental line with aplant homozygously resistant to downy mildew of onion according to theinvention. Said plant is for example obtainable by carrying out themethod of the invention as disclosed above.

The Allium cepa parental line may be any line of the species Alliumcepa, it is preferably a line well described and known forcharacteristics of interest. For example it is a cultivar line known forits taste, fast growing or other agronomical important traits.

According to a preferred embodiment, the parental line is a cytoplasmicmale sterile Allium cepa inbred line.

Any plant of the invention which has been described is, according to apreferred embodiment of the invention, a cultivated onion or shallot.

LEGEND OF THE FIGURES

FIG. 1: This figure illustrates schematically a potential way oftransmission of Pd resistance locus and lethal sequences to the progenyof self-pollination for 4 different resistant Allium. The right columngives the percentage of resistance in progeny and is indicative ofwhether the parent plant has at least a homologue of chromosome 3 whichbears the resistance locus without the lethal sequences.

FIG. 2: this figure depicts the GISH karyotypes for the 14 downy mildewresistant plants from population 2348, which have been analysedaccording to the protocol described in example 2. 2348-1, 2348-3,2348-4, 2348-5, 2348-6, 2348-7, 2348-8, 2348-11, 2348-13, 2348-14,2348-15, 2348-16, 2348-18, and 2348-19 are different plants from thesame parental line 2348.

FIG. 3: this figure depicts the GISH karyotypes for the 6 downy mildewresistant plants from population 3591, which have been analysedaccording to the protocol described in example 2. 3591-1 (FIG. 3A),3591-3 (FIG. 3B), 3591-4 (FIG. 3C), 3591-5 (FIG. 3D), 3591-6 (FIG. 3E)and 3591-8 (FIG. 3F) are different plants from the same parental line3591.

EXAMPLES Example 1 Obtaining of a Homozygously Resistant Line of Alliumcepa Introduction:

Downy mildew in onion is caused by the fungus Peronospora destructor(Berk.) Casp. (Mukerji, ref 5). It is an obligate pathogen that can onlybe maintained on plant tissue, not on agar or liquid media in vitro.

Tests on disease resistance can be carried out in various ways, see forexample de Vries et al (ref 3). Spontaneous epidemics may occur in thefield, if conditions are favourable for the disease. Artificial testscan be carried out in greenhouses or climate rooms under more controlledenvironments. Both young plants and sprouting bulbs can be tested. Thesymptoms are yellowing of the leaves and grey sporulation.

In the breeding program followed by the inventors, various methods havebeen applied during the process of backcrossing and during the processof selfing in order to obtain homozygous lines.

This report describes the trials which resulted in the conclusion thatthe resistance did not inherit as simply as expected: monogenicdominant, and which resulted in the production of the line 3591 of theinvention, with complete resistance to downy mildew.

Materials and Methods.

The starting point of this study is the plant called F₁BC₂ disclosed inde Vries et al (ref 3). This plant has been obtained by a firstinterspecific cross between Allium cepa and Allium roylei (F1), followedby two successive backcrosses to Allium cepa (BC2). The presentinventors have continued to carry out backcrosses of said plant materialto their breeding lines and they have carried out selfings of the BC5generation in order to obtain homozygous lines. S1, S2 and S3 indicatesrespectively that 1, 2 or 3 successive selfing (self-pollination) stepshave been carried out.

A first field test was carried out with F3 lines (F₁BC₅S₂), derived fromselfing resistant plants of different F2 populations (F₁BC₅S₁). Plantswere sown out on the main breeding field of the inventors.

Spreader bulbs that were previously inoculated in the greenhouse wereplaced in the field (Hildebrand et al, ref 7).

Inoculum was made by suspending conidiospores in demi-water. Leaves withspores were washed off with demi-water over two layers of cheese cloth.The suspension was diluted to 5.0×10⁴ spores/ml. The spores wereinoculated into bulbs, from the bulb equator to the basal plate, with asyringe with needle. The bulbs were placed in plastic bags with somehumid paper during 24 h at 10° C. After that the temperature was raisedto 15° C. in order to stimulate the growth of mycelium. Then the bulbswere planted out in the field.

An epidemic started and during the second half of August infected plantswere counted. During the growing season and until the stage of fall overof the leaves and ripening of the bulbs, counts were made ofnon-infected bulbs in order to estimate the resistance score andsegregation ratio of the different lines. Resistant plants do not showsymptoms at all. If any lesions could be scored, the plant wasconsidered as susceptible.

In second field test, 353 plots were drilled on the breeding field, inthe first week of April.

The field was surrounded by bulbs, in order to stimulate the developmentof the downy mildew epidemic. A heavy infection started which resultedin a 100% infection of control plots of the susceptible varietyStaccato. Counts were made of infected and non-infected plants. Bulbs ofrelevant lines were harvested and used for further research and seedproduction for the year after.

Results.

The first field test resulted in heavy infection of the onion plants. Aslong as the symptoms could be scored reliably, counts were made of thesusceptible plants. Some early lines fell over in an early stage and nodistinction could be made between dying of the leaves and downy mildewsymptoms. For these lines only the susceptible plants were scored andnot the resistant ones, because the resistant class would in these earlylines contain many escapes. In total there were 20 lines. 34 lines werecounted for both susceptible and resistant plants and these lines werefalling over much later and the scoring on resistance could be donereliably. Data are given in table 1.

The second field test again gave a high downy mildew infection. Specialcontrol plots of the susceptible variety Staccato were 100% infected. Inthis field test of 353 different breeding lines, only one number wasfound that gave no infected plants. This breeding line 4018282 sownunder the plot number 3591 gave 140 plants not infected. 21 other F4lines (F₁BC₅S₃), derived by selfing from the same F3 line, 3997284,showed segregation for resistance, table 2.

TABLE 1 Downy mildew field test n°1: Breeding line Percentage PlantsPlants F3 S2 Resistance susceptible resistant 3977304 72.6   63  1673977305 67.5  124  258 3977307 90.7   26  254 3977311 52.9  177  1993977313 57.7  224  305 3977319 55.6  248  311 3977322 51.7  357  3823977326 50.9   82   85 3977328 59.1   56   81 3977333 57.1  275  3663977334 62.5   48   80 3977336 66.7   25   50 3977337 56.2  312  4013977338 70.3  33   78 3977343 74.0   20   57 3977344 68.4   24   523977350 58.3   43   60 3977351 53.1   38   43 3977357 73.2   56  1533977358 92.5    7   86 3977367 68.3   33   71 3977375 60.9   88  1373977377 71.0   54  132 3977378 57.4  103  139 3977379 56.0   80  1023977380 61.7   75  121 3977381 66.5   56  111 3977382 58.9  304  4363977385 66.3   31   61 3977387 60.2   37   56 3977392 55.9  152  1933977394 72.1   39  101 3977395 55.0   45   55 3977396 47.6   66   60Total 60.7 3401 5243

TABLE 2 Downy mildew field test n°2: Breeding line Percentage PlantsPlants F4 S3 Resistance susceptible resistant 4018268 58.3 63  884018269 61.5 82 131 4018270 69.2 66 148 4018272 61.7 57  92 4018273 64.560 109 4018274 75.1 46 139 4018275 71 67 164 4018276 68.5 78 170 401827863.7 85 149 4018279 56.9 97 128 4018280 66.9 46  93 4018281 60.5 90 1384018282 100  0 140 4018283 62.2 56  92 4018284 66.7 47  94 4018287 54.884 102 4018288 71.4 46 115 4018289 71.5 55 138 4018290 53.8 55  644018291 67.8 38  80 4018292 65.2 64 120 4018293 72.2 80 208

Discussion.

The field trial n°1 gave the first clear indication that breeding fordowny mildew resistant onions was not as simple as previously expected.In case of a monogenic, dominantly inherited resistance, one wouldnormally expect to find at least 1/3 of the lines being fully resistant,after selfing resistant F2-plants. Only RR plants give a fully resistantprogeny, Rr plants will segregate, and in a F2 after selection forresistance RR plants comprise 1/3 of the population (where “R” standsfor allele bearing the dominant resistance gene, “r” for the recessivesusceptible gene).

54 lines were tested and 34 could reliably be scored for both resistanceand susceptibility, but all of the 54 lines segregated. This means aratio significantly different from 1/3.

Total ratio of resistant plants over all lines was lower than expected(60,65%) If a heterozygous plant was selfed, a resistant ratio of 75%was expected in case of a single dominant gene.

Individual lines, like 3977307 and 3977358 gave a different ratio. Thismay be caused by escapes. Inbred lines derived from 3977303 have beentested, and all segregated, with ratios varying between 52% and 73%(data not shown). Plants that failed to be infected are classified asresistant, but escaped to show symptoms, maybe due to early decay offoliage, low population, that leads to a less humid micro-climate orother disease or trips infections.

The field test n°2 resulted in the discovery of the 3591-plot,containing line 4018282. This line did not show susceptible plants in aheavy infected field. The field contained 353 lines which confirmed thecomplexity of the inheritance. Otherwise a much higher frequency ofhomozygous lines was found and not only line 4018282/3591.

Out of plot 3591 plants were taken into the greenhouse for research onthe A. roylei-introgression fragment to be carried out (see example 3)and for seed production purposes. The results presented in example 3showed that 3591 contained a smaller introgression fragment. Some of theplants are heterozygous for the small and large introgression fragmentsand plant 3591-1 was homozygous for the small introgression fragment.

The uniqueness of this line is illustrated by the results of the sisterlines given in table 2. These lines have all been derived from the sameF3 line by selfing and none of them show homozygosity.

Example 2 Genomic In situ Hybridization (GISH) with the Breeding Line2348, Generation F₁BC₅S₃ (breeding line 2348 from sample number 4008191of plot 2348 gave a 66% resistance in the field)

The technique of Genomic In situ Hybridization (GISH) has been developedin onion by Khrustaleva and Kik (ref 9 and 10) and has been used in thepresent invention in order to distinguish the genomic Allium royleiinserts/segments from the Allium cepa chromosomes. This technique thusallows the visualization of the introgression fragments from Alliumroylei in the genome of an Allium cepa plant.

A first set of experiments were carried out by Plant ResearchInternational BV, Wageningen (NL), on resistant plants, howeversegregating, onion plants, which correspond to 14 plants F₁BC₅S₃,wherein F₁ is the interspecific hybrid between Allium roylei and Alliumcepa, BC₅ indicates 5 successive backcrosses and S₃ 3 successiveself-pollinations.

Introduction:

GISH is a powerful technique for detection of the introgression ofchromatin material from one species onto another species. The advantageof GISH is that the introgression process is visualized by means of‘pictures of the introgressed genome’. With this technique, it is alsopossible to establish if a particular region of the genome is homozygousor heterozygous, thanks to the use of molecular cytogenetic markerswhich are co-dominant. By this technique, it is also possible todetermine in which chromosome an introgressed gene of interest ispresent.

Plant Material:

The starting material is the breeding line 2348, generation F₁BC₅S₃.Young root tips are collected and up to 100 slides are made formetaphase spread analysis (for method description, see Khrustaleva andKik, (ref 9 and 10)). The slides with well spread metaphases and whichcontain a full set of chromosomes (2n=2x=16) are selected for GISHexperiments.

Method:

Genomic DNA is extracted from young leaves of A. cepa and A. royleiusing the CTAB method of Rogers and Bendich (ref 13).

Genomic DNA of A. roylei is labeled with Dig-11-dUTP(Digoxigenin-11-2′-deoxy-uridine-5′-triphosphate) by a standardnick-translation protocol (Boehringer, Manheim, Germany). Genomic DNA ofA. cepa is used as a block DNA. In situ hybridization, immunologicaldetection and microscopy procedure are as previously described byKhrustaleva and Kik, (ref 9 and 10). Karyotype analyses are carried outaccording to the standard onion nomenclature system proposed by Kalkman(ref 12) and confirmed by the Fourth Eucarpia Allium Symposium by deVries (ref 8). The chromosome measurements from 3-5 metaphases per eachaccession are performed with a free software programme for Windows fromColorado State University:(http://www.colostate.edu/Depts/biolog/MicroMeasure).

Results:

GISH analysis of 14 resistant plants from the breeding line 2348,generation F₁BC₅S₃ showed that all plants possess the A. roylei segmentharbouring the gene(s) for downy mildew resistance (FIGS. 2A, 2B and2C). Karyotype analysis revealed that the recombinant chromosome whichcarried the A. roylei segment is chromosome 3 (centromere index: 41.7;relative chromosome length: 13.8). Thirteen plants did possess only oneA. roylei segment on chromosome 3, the other homologous chromosomesproved to be composed of only A. cepa chromatin. Without any doubt,these results proved that all 13 plants of breeding line 2348 areheterozygous for the segment containing the downy mildew resistancegene(s). Accession 2348-5 possesses two A. roylei segments: one on thelong arm of chromosome 3 that carried the downy mildew resistancegene(s) and an additional one on the long arm of chromosome 4(centromeric index: 39.3; relative chromosome length: 12.6). Bothsegments are heterozygously present in the genome.

GISH analysis allows also the size determination of the introgressedsegment on the long arm of chromosome 3 from A. roylei into the A. cepachromosome. The segment on average is 25.2% of the entire chromosomelength and 43.9% of the long arm length.

Discussion:

By this method, the introgression of A. roylei chromatin materialcarrying downy mildew resistance genes into onion genome has beensuccessfully detected. The present data proves the heterozygous natureof the downy mildew resistance gene(s) in breeding line 2348, generationF₁BC₅S₃. In all 14 individuals selected as resistant in the field, wasfound an A. roylei segment in only one homologue of chromosome 3. Theaforementioned results could be explained by hypothesizing that a gene(or lethality factor) which is necessary for the A. cepa development,for example for seed development, and located near the downy mildewresistance gene(s), is responsible for the absence of homozygous plants.Indeed, It may be supposed that the replacement of an essential A. cepagene by the corresponding A. roylei gene during introgression leads to asevere disturbance of the plant development, for example seeddevelopment, when placed in an A. cepa cytoplasm background. A viablehomozygous resistant onion can be obtained only if recombination occursbetween the downy mildew resistance gene(s) and the lethality factor,should this be possible.

During backcrossing to A. cepa and selection for downy mildewresistance, only those plants containing the A. roylei segmentharbouring the downy mildew resistance gene(s) will be maintained. Thisleads eventually to plants, which consist only of A. cepa chromatin anda segment of A. roylei. However, according the above-mentionedhypothesis, this segment has replaced the A. cepa gene necessary fordevelopment. In a heterozygous condition, seeds develop and plants willbe obtained, but in homozygous condition no plants are formed.

Example 3 Genomic in situ Hybridization with the Breeding Line 3591,Generation F₁BC₅S₃ Introduction:

Following the analysis of 14 downy mildew resistant plants from breedingline 2348, generation F₁BC₅S₃, it was shown that all plants wereheterozygous for the Pd resistance locus. The A. roylei segmentharbouring the gene(s) for downy mildew resistance was detected only onone of the homologues of chromosome 3, the other homologue consistingentirely of A. cepa chromatin. This further analysis concerns thebreeding line 3591 in which no segregation takes place for downy mildewresistance in the fields. In view of this, it Is expected to find inthis population, via GISH, plants which are homozygous for the area ofchromosome 3 which harbours the downy mildew resistance locus.

Plant Material:

From 6 individual plants, breeding line 3591, generation F₁BC_(S)S₃,young root tips are collected and up to 50 slides are made for metaphasespread analysis (for method description, see Khrustaleva and Kik, ref9). The slides with well spread metaphases and which contain a full setof chromosomes (2n=2x=16) are selected for GISH experiments.

Methods:

Details are given under section “method” of the previous example.

Results:

Plant Research International BV, Wageningen, NL carried out GISHanalyses of 6 resistant plants from the breeding line 3591, generationF₁BC₅S₃ which showed that all plants are homozygous A. roylei for thearea harboring the downy mildew resistance locus (see FIGS. 3A to 3F).Karyotype analysis reveals the presence of an A. roylei segment in bothhomologues of chromosome 3 (centromere index: 41.7; relative chromosomelength: 13.8). Amongst 6 analyzed plants, accession 3591-1 possesses 2small segments on both homologues of chromosome 3 (FIG. 3A). The size ofthe A. roylei introgressed segments is the same on both homologues andaverages: 17.9±0.78% of the length of the long arm. Five plants, namelyaccession number 3591-3, 3591-4, 3591-5, 3591-6 and 3591-8, possess twoA. roylei introgressed segments that differed in size. (FIGS. 3B, 3C,3D, 3E and 3F) The size of the larger segment averages 42.8±1.09% of thelength of the long arm and the size of smaller one averages 17.9±0.78%.

Discussion:

The present results confirm the hypothesis put forward to explain theheterozygous nature of the downy mildew resistance locus in breedingline 2348. It might be indeed hypothesized that a lethality factor,deleterious for plant development and which is located near the downymildew resistance gene(s), is responsible for the absence of homozygousresistant plants, or hypothesized that an essential A. cepa gene,necessary for plant development, is knocked out by the introgression andis responsible for the absence of homozygous resistant plants.

It is supposed that the A. roylei introgression segment possesses alsothe corresponding gene that does not function in an exclusive A. cepacytoplasm (i.e. nucleo-cytoplasmic interaction). In a homozygous A.roylei condition, the essential A. cepa gene will be fully replaced bythe corresponding A. roylei gene, consequently it will give no viableseeds. Only if recombination occurs between the downy mildew resistancegene(s) and the essential gene (or lethality factor), a viablehomozygous resistant plant will be obtained.

GISH analysis of breeding line 3591 proves that a homozygous resistantplant (3591-1) can be obtained due to a recombination event between theresistance gene and the lethality factor. The five other accessionspossess both small and large A. roylei introgression segments, whichmeans that these plants are homozygous for the resistance gene(s) andheterozygous for the A. roylei lethality factor.

It must be noted that, as expected from the hypothesis mentioned above,no plants possessing two large A. roylei introgression segments havebeen found, because this genetic constitution results in no seeddevelopment.

Example 4 Identification of Markers Linked to the Peronospora destructorResistance Locus (Derived form A. roylei).

Plant material (number of individuals):

-   1× Allium roylei (homozygous resistant)-   4× (named AcA to AcD) and 24× (named Ac01 to Ac24) Allium cepa    (homozygous susceptible)-   1× 3591-1 (homozygous resistant; small introgression fragments)-   1× 3591-3 (homozygous resistant, small and large introgression    fragments)-   14× 2348-* (heterozygous resistant; large fragment).

Introduction:

For complement information on the AFLP™ technique, reference is made tothe publication referenced under n°4.

The aim of this example is to identify AFLP™ markers linked to thePeronospora destructor resistance locus in onion. The resistance locusis derived from Allium roylei.

As shown in example 2, a large introgression fragment has beenidentified in some individuals (individuals 2348-*). Besides theseindividuals, also two individuals were obtained which possess a smallerintrogression fragment (3591-1; small fragment, and 3591-3; small andlarge fragments). To identify markers associated with the resistancelocus, and especially obtain markers which are located on the smallerintrogression fragment, an adapted Bulked Segregant Analysis (BSA, seeMichelmore, ref 11) strategy was carried out on four individuals/pools:

-   I: individual 2348-6; heterozygous resistant; large introgression    fragment,-   II: individual 3591-1; homozygous resistant; small introgression    fragment,-   III: individual Allium roylei; homozygous resistant,-   IV: a pool of four A. cepa individuals (AcA to AcD); homozygous    susceptible.

The four separate Allium cepa individuals, 13 remaining ‘2348’individuals and individual 3591-3 were used for verification of thelinked markers. Markers which were found to be linked to the trait ofinterest are finally checked on a set of 24 Allium cepa individuals(Ac01 to Ac24) to identify false positive markers.

Results:

Biological material: DNA was extracted from leaf material of 31individuals (28× Allium cepa and individuals 3591-1, 3591-3 and Alliumroylei) and DNA of the 14× ‘2348’ individuals and for all individualsPstI/MseI templates were generated. No reliable AFLP™ fingerprints couldbe generated for the individuals 2348-4, 2348-5, 2348-11 and 3591-3,therefore these individuals were excluded from further analysis.

Marker Identification and Verification:

An adapted BSA approach was carried out on four individuals or pool I,II, III, IV as described above.

The BSA was carried out using 96 primer combinations in thePstI+3/MseI+3 matrix, an overview of the primer combinations used isgiven in table 3 and table 6 gives the nomenclature of AFLP™ primerenzyme combinations.

The BSA resulted in the identifications of 34 candidate markers of whicheight candidate markers are possibly linked to the small introgressionfragment. From these 34 candidate markers, a selection was made of 13markers including the eight markers that are possibly linked to thesmall introgression fragment. This selection of 13 markers was used forverification on more individuals. For the verification four Allium cepaindividuals, the 13 remaining ‘2348’ individuals and individual 3591-3were used.

TABLE 3 Primer combination used for the BSA approach. M32 M34 M35 M37M47 M48 M50 M51 M54 M59 M60 M62 P31 X X X X X X X X X X X X P32 X X X XX X X X P33 X X X X P38 X X X X X X X X P39 X X X X X X X X X X X X P35X X X X X X X X X X X X P42 X X X X X X X X P43 X X X X X X X X X X X XP44 X X X X X X X X P45 X X X X X X X X X X X X

On basis of the verification, three candidate markers (of which two arepossibly linked to the small introgression fragment) did not show aclear linkage with the trait of Interest. However a total of fourrespectively six candidate markers, showed a clear linkage with thelarge respectively small introgression fragment. However two of thesecandidate markers, linked to the small introgression fragment are alsopresent in one of the four Allium cepa individuals. Therefore thesemarkers are found to be not useful for further analysis. The results ofthe verification are shown In Table 4.

Markers P32/M62-061, P33/M32-151, P35/M51-330 and P43/M35-190 are foundto be linked to the small introgression fragment.

Extra-verification on 24 Allium cepa individuals:

To overcome false positive markers the four candidate markers(P32/M62-061, P33/M32-151, P351M51-330 and P43/M35-190) linked to thesmall introgression fragment were checked on 24 extra Allium cepaindividuals (Ac01 to Ac24). The obtained marker scores are in accordancewith the expected score (see table 5).

TABLE 4 Overview of marker scores on individual plants Markers linked tolarge introgression fragment Markers linked to small introgressionfragment P31/ P32/ P32/ P33/ P33/ P45/ P32/ P33/ P35/ P43/ M48-186M62-322 M62-079 M32-115 M32-155 M35-062 M62-061 M32-151 M51-330 M35-1902348-1 + + + + + + + + + + 2348-3 + + + + + + + + + + 2348-4 x x x x x xx x x x 2348-5 x x x x x x x x x x 2348-6 + + + + + + + + + +2348-7 + + + + + + + + + + 2348-8 + + + + + + + + + + 2348-11 x x x x xx x x x x 2348-13 + + + + + + + + + + 2348-14 + + + + + + + ? + +2348-15 + + ? + + + + + + + 2348-16 + + + + + + + + + +2348-18 + + + + + + + + + + 2348-19 − + + + + x + − + + 3591-3 x x x x xx x x x x 3591-1 − − − − + + + + + + A. roylei + + + + + + + + + + A.cepa A − − − − − − − − − − A. cepa B − − − − − − − − − − A. cepa C − − −− − − − − − − A. cepa D − − − − + + − − − − + = AFLP ™ fragment present,− = AFLP ™ fragment absent, x = no good product generated, ? = notscorable.

Discussion and Conclusion:

To identify markers associated with the Peronospora destructorresistance locus in onion, and especially to obtain markers which arelocated on the smaller introgression fragment, an adapted BulkedSegregant Analysis (BSA) strategy was carried out on fourindividuals/pools. The BSA was carried out using a total of 96 PstI/MseIprimer combinations (which were screened on four individuals/pools).

TABLE 5 Overview of marker scores on individual plants Markers linked tolarge introgression fragment Markers linked to small introgressionfragment P31/ P32/ P32/ P33/ P33/ P45/ P32/ P33/ P35/ P43/ M48-186M62-322 M62-079 M32-115 M32-155 M35-062 M62-061 M32-151 M51-330 M35-190A. cepa 1 − − − − A. cepa 2 − − − − A. cepa 3 − − − − A. cepa 4 − − − −A. cepa 5 − − − − A. cepa 6 − − − − A. cepa 7 − − − − A. cepa 8 − − − −A. cepa 9 − − − − A. cepa 10 − − − − A. cepa 11 − − − − A. cepa 12 − − −− A. cepa 13 − − − − A. cepa 14 − − − − A. cepa 15 − − − − A. cepa 16 −− − − A. cepa 17 − − − − A. cepa 18 − − − − A. cepa 19 − − − − A. cepa20 − − − − A. cepa 21 − − − − A. cepa 22 − − − − A. cepa 23 x − x − A.cepa 24 − − − − + = AFLP ™ fragment present, − = AFLP ™ fragment absent,x = no good product generated, ? = not scorable.

The BSA resulted in the identification of 34 candidate markers of which8 candidate markers were possibly positioned in the small introgressionfragment. After verification, a total of four markers were identifiedwhich are located in the small introgression fragment (on which also thePeronospora destructor resistance locus is located).

To overcome false positive markers, the four candidate markers(P32/M62-061, P33/M32-151, P35/M51-330 and P43/M35-190) linked to thesmall introgression fragment were checked on 24 extra Allium cepaindividuals. The obtained marker scores are in accordance with theexpected score. Therefore, these markers are found to be linked to thesmall introgression fragment of interest and can be used for furtherselection procedures.

TABLE 6 Nomenclature of AFLP ™ primer enzyme combination exten- primerexten- primer exten- primer sion code sion code sion code AAA 31 CCC 52GGG 73 AAC 32 CCG 53 GGT 74 AAG 33 CCT 54 GTA 75 AAT 34 CGA 55 GTC 76ACA 35 CGC 56 GTG 77 ACC 36 CGG 57 GTT 78 ACG 37 CGT 58 TAA 79 ACT 38CTA 59 TAC 80 AGA 39 CTC 60 TAG 81 AGC 40 CTG 61 TAT 82 AGG 41 CTT 62TCA 83 AGT 42 GAA 63 TCC 84 ATA 43 GAC 64 TCG 85 ATC 44 GAG 65 TCT 86ATG 45 GAT 66 TGA 87 ATT 46 GCA 67 TGC 88 CAA 47 GCC 68 TGG 89 CAC 48GCG 69 TGT 90 CAG 49 GCT 70 TTA 91 CAT 50 GGA 71 TTC 92 CCA 51 GGC 72TTG 93 GGG 73 TTT 94

Example 5 Determination of the Presence, in a Plant of Allium Genus, ofan Introgression Fragment of A. roylei Conferring Resistance to DownyMildew of Onion, Which Can be Present Homozygously Without CausingLethality

In a first step, the presence of Pd resistance locus to downy mildew ofonion is assayed in the plant under examination. The presence of saidlocus can be assayed using AFLP™ technique, as exemplified in thepreceding example or by assaying the resistance of the plant to naturalor artificial inoculations by Peronospora destructor.

If the plant possesses in its genome a Pd resistance locus, thehomologues of chromosome 3 of said plant necessarily correspond to oneof the 4 possibilities depicted in FIG. 1 (with and without linkage tolethal sequences).

In order to distinguish the first three genomes, which are part of thepresent invention, from the fourth, self-pollination of the plant underexamination is carried out. The progeny of the self-pollination is thenexamined for the presence of the Pd resistance locus. As can been seenfrom FIG. 1, only the first three genomes give a progeny which istheoretically at least 75% resistant, whereas the fourth depictedgenome, which is not part of the present invention, gives a progenywhich is less than 67% resistant to downy mildew of onion.

Knowing the percentage of resistant progeny after self-pollination, itis thus possible to deduce whether the tested plant is in one of thefirst three categories depicted or is in the fourth, i.e. whether or notit is in the scope of the present invention.

Example 6

Plants from the line 3591, similar to 3591-1 in the respect that theyare all homozygous resistant, and having the two small introgressionfragments bearing the resistance locus are crossed with a hybrid in athree way breeding scheme (wherein the three way cross has a singlecross as the female parent and the plants from the 3591 line as the maleparent).

The hybrid resulting from this cross, 37-1001 has been trialed infields. This hybrid is a mid-early extreme long day type of onion,suitable for mid long storage. It shows a medium strong foliage vigour,a very uniform fall over, uniform round-flat round shaped bulbs (3.8units/he.), a good yield potential, good skins with yellow-brown colour,a good firmness (3.4 mm). Field test data are shown in table 7,comparing 37-1001 with Tasco and Drago, two commercialized onion hybridsfrom Nickerson Zwaan. As can be seen from this table, the features ofagronomical interest are similar in all plants.

Most important to the present invention, in disease field tests held for2 years consecutively in 2 replications, all the tested plants 37-1001are resistant, whereas a variety used as susceptible check, Staccato(Nickerson Zwaan hybrid), has 100% plants infected with downy mildew.

TABLE 7 Leaf vigour Leaf tips Number Relative Erectness (1 = weak (1 =yellow of bolters earliness Leaf colour of leaves growing or brown (=plants % of bolters (measured (1 = light (1 = leaves, 5 = leaf tips, 5 =producing (on total via timing Yield (per green, 5 = horizontal,vigourous healthy green a flower number of fall-over square Name darkgreen) 5 = upright) leaves) leaf tips) stem) of plants) of leaves)meter) Tasco 3 3 5 3 4 1.0 131.8 8.883 Tasco 3 3 5 3 7 1.8 115.5 8.610Tasco 3 3 5 3 10 2.8 113.7 8.467 Tasco 3 3 4 3 2 0.6 133.6 8.028Average: 3 3 5 3 6 2 124 8.50 Tasco Drago 4 3 4 3 1 0.3 97.5 7.640 drago3 3 3 3 3 0.8 113.7 7.971 drago 5 3 4 3 3 0.8 79.4 7.694 drago 4 3 5 3 71.9 115.5 8.126 Average: 3 3 5 3 4 1 106 7.99 Drago 37-1001 3 3 4 3 0.0115.5 8.657 37-1001 3 3 4 3 1 0.3 97.5 8.622 37-1001 3 3 4 3 2 0.5 113.78.114 37-1001 3 3 5 3 0.0 115.5 8.852 Average: 3 3 5 3 2 0.4 109.6 8.44637-1001 Skin quality Firmness (1 = no (= mm of papery skins deformationNumber Relative Relative around bulb, when of rings yield Average % of %of skin 9 = a lot compressed, counted (100 = trial bulb weight skinlessretaining of papery so the lower when bulb Name average) (g) bulbs bulbsskins) the better) is cut Tasco 106.6 130 10.6 92.9 6 3.61 4.96 Tasco103.4 122 6.0 97.7 6 3.62 5.24 Tasco 101.6 128 4.9 98.8 7 3.51 5.32Tasco 96.4 124 12.3 91.1 6 3.60 4.08 Average: 102 126 8 95 6 4 5 TascoDrago 91.7 119 4.5 99.2 7 3.38 5.16 drago 95.7 111 5.4 98.3 6 2.93 4.42drago 92.4 118 3.3 100.5 7 3.08 4.63 drago 97.6 125 6.8 96.9 6 3.26 4.68Average: 96 120 6 98 6 3 5 Drago 37-1001 103.9 129 13.6 89.8 5 3.57 4.2037-1001 103.5 124 6.3 97.4 6 3.17 4.36 37-1001 97.4 117 8.3 95.3 6 3.194.46 37-1001 106.3 122 11.8 91.7 6 3.22 4.92 Average: 101.4 122 9.1 94.46 3.28 4.54 37-1001

Example 7

In the following, line 3591 or plants 3591 are homozygous resistantplants bearing the small introgression fragment, they are all plantssimilar to 3591-1 because they also contain the small introgressionfragment in a homozygous condition and belong to the same breeding line.

3591 plants have also been used successfully as starting material forthe development of new parental breeding lines useful in producing newcommercial onion hybrids, resistant to the downy mildew of onion. The3591 plants are first crossed with an inbred onion line to produce an F1hybrid.

Indeed, through backcrossing to other pollinator lines, the downy mildewresistance gene can be introduced into other pollinator lines of manydifferent onion types.

A first cross is made between a resistant plant having the two smallintrogression fragments bearing the resistance locus and a susceptibleline. This cross gives rise to hybrids (F1s) which contain the smallintrogression fragment heterozygously. These heterozygously resistant F1plants are used for subsequent backcrossing with a susceptible line orthe susceptible line used to produce the F1 resistant plants. Suchbackcross step will give rise to a population which has 50% resistantand 50% susceptible plants. This 1:1 segregation will occur with everyback-cross generation due to the inheritance of the dominant Pdresistance locus. All susceptible plants can be phenotyped for downymildew susceptibility and eliminated. New onion inbred lines usable asparent material for producing F1 hybrids are obtained through severalsteps of backcrossing followed by self pollination as known by a manskilled in the art.

This inheritance is confirmed by the field data; among the nine plantsproduced by the cross sy108a*3591, the nine plants are resistant.Similarly, among the four plants produced by the cross fra*3591, allfour plants are resistant. Also similarly all the 3 plants produced fromthe cross syt*3591 are resistant to the downy mildew of onion. Theseplants are then backcrossed as mentioned hereunder.

Example 8

All plants can also be genotyped with the markers disclosed in thepresent invention; among the 16 F1 hybrids produced when crossing 3591plants having the two small introgression fragments bearing theresistance locus with various other inbred parental lines, all showedthe expected 61 nucleotide fragment when the pair of primers (A) wasused (marker P32/M62-061 linked to the small introgression fragment) anddid not show the 79 nucleotide fragment usually highlighting thepresence of the large introgression fragment when the marker P32/M62-079is used (the pair of primers (B′)).

REFERENCES

-   1. Meer, Q. P. van der and Vries, J. N. De. An interspecific cross    between Allium roylei Stearn and Allium cepa L., and its backcross    to A. cepa. Euphytica 47:29-31, 1990.-   2. Kofoet, A. et al. Inheritance of resistance to Downy Mildew    (Peronospora destructor [Berk.] Casp.) from Allium roylei Stearn in    the Backcross Allium cepa L.* (A. roylei* A. cepa). Plant Breeding    105:144-149, 1990.-   3. Vries, J. N. De et al. Linkage of downy mildew resistance genes    Pd1 and Pd2 from Allium roylei Stearn in progeny of its    interspecific hybrid with onion (A. cepa L.). Euphytica 64:131-137,    1992.-   4. Vos, P. et al. AFLP: a new technique for DNA fingerprinting.    Nucleic Acids Research. Vol 21, 21: 4407-4414, 1995.-   5. Mukerji, K. G. Peronospora destructor. CMI Description of    Pathogenic fungi and Bacteria No456, 1975.-   6. Viranyi, F. Downy Mildew of Onion. D M Spencer, The Downy    Mildews. Chap. 21:461-471, 1981.-   7. Hildebrand, P. D., and Sutton, J. C. Maintenance of Peronospora    destructor in onions sets. Canadian Journal of Plant Pathology    2:239-240, 1980.-   8. Vries, J. N. De. Onion chromosome nomenclature and homoeology    relationships—workshop report. Euphytica 49:1-3, 1990.-   9. Khrustaleva, L. I. and Kik, C. Cytogenetical studies in the    bridge cross Allium cepa * (A. fistulosum * A. roylei. Theor. Appl.    Genet. 96: 8-14, 1998.-   10. Khrustaleva, L. I. and Kik, C. Introgression of Allium    fistulosum into A. cepa mediated by A. roylei. Theor. Appl. Genet.    100: 17-26, 2000.-   11. Michelmore, R. W. et al. Identification of markers linked to    disease-resistance genes by bulked segregant analysis: A rapid    method to detect markers in specific genomic regions by using    segregating populations. Proc. Natl. Acad. Sci. USA. 88: 9828-9832,    1991.-   12. Kalkman, E. R. Analysis of the C-banded karyotype of Allium    cepa L. Standard system of nomenclature and polymorphism. Genetica    65:141-148, 1984.-   13. Rogers and Bendich. In: Gelvin S B, Schilperoort R A (eds) Plant    Molecular Biology manual A6, Kluwer Academic Publ., Dordrecht, the    Netherlands; 1-10, 1998.

1.-11. (canceled)
 12. An onion or shallot plant of Allium cepa specieswhich is resistant to downy mildew of onion caused by the fungusPeronospora destructor (Pd), which plant comprises a Pd resistance locuspresent homozygously in the long arm of chromosome 3 in the genome ofsaid plant, the Pd resistance locus having a sequence identical to agenomic segment of allium roylei conferring resistance to downy mildewin A. roylei, wherein the presence of the Pd resistance locus ischaracterized by a 61 nucleotide amplification product with theselective amplification primers (A) 5′-GACTGCGTACATGCAGAAC (SEQ ID NO:5)and 5′ GATGAGTCCTGAGTAACTT (SEQ ID NO:6) in the genome of said plantrestricted with restriction enzymes PstI and MseI; and ligated with thefollowing oligonucleotide adapters: alpha 5′-CTCGTAGACTGCGTACATGCA(SEQ ID NO: 1) CATCTGACGCATGT-5′, (SEQ ID NO: 2) and beta5′-GACGATGAGTCCTGAG (SEQ ID NO: 3) TACTCAGGACTCAT-5′ (SEQ ID NO: 4)


13. An onion or shallot plant of Allium cepa species which is resistantto downy mildew of onion caused by the fungus Peronospora destructor(Pd), which plant comprises a Pd resistance locus present homozygouslyin the long arm of chromosome 3 in the genome of said plant, the Pdresistance locus having a sequence identical to a genomic segment ofAllium roylei conferring resistance to downy mildew in A. roylei,wherein the presence of the Pd resistance locus is characterized by a151 nucleotide amplification product with the selective amplificationprimers (B) 5′-GACTGCGTACATGCAGAAG (SEQ ID NO: 7) and5′-GATGAGTCCTGAGTAAAAC (SEQ ID NO: 8)

in the genome of said plant restricted with restriction enzymes PstI andMseI; and ligated with the following oligonucleotide adapters: alpha5′-CTCGTAGACTGCGTACATGCA (SEQ ID NO: 1) CATCTGACGCATGT-5′,(SEQ ID NO: 2) and beta 5′-GACGATGAGTCCTGAG (SEQ ID NO: 3)TACTCAGGACTCAT-5′ (SEQ ID NO: 4)


14. An onion or shallot plant of Allium cepa which is resistant to downymildew of onion caused by the fungus Peronospora destructor (Pd), whichplant comprises a Pd resistance locus present homozygously in the longarm of chromosome 3 in the genome of said plant, the Pd resistance locusbeing on a genomic Allium roylei insert, wherein the presence of the Pdresistance locus is characterized by a 330 nucleotide amplificationproduct with the selective amplification primers (C)5′-GACTGCGTACATGCAGACA (SEQ ID NO: 9) AND 5′-GATGAGTCCTGAGTAACCA(SEQ ID NO: 10)

in the genome of said plant restricted with the restriction enzymes PstIand MseI; and ligated with the following oligonucleotide adapters: alpha5′-CTCGTAGACTGCGTACATGCA (SEQ ID NO: 1) CATCTGACGCATGT-5′,(SEQ ID NO: 2) and beta 5′-GACGATGAGTCCTGAG (SEQ ID NO: 3)TACTCAGGACTCAT-5′ (SEQ ID NO: 4)

15.-28. (canceled)
 29. An onion or shallot plant of Allium cepa which isresistant to downy mildew of onion caused by the fungus Peronosporadestructor (Pd), which plant comprises a Pd resistance locus presenthomozygously in the long arm of chromosome 3 in the genome of saidplant, the Pd resistance locus being on a genomic Allium roylei insert,wherein said genomic Allium roylei insert is characterized by thepresence of: a. a 61 nucleotide amplification product with the primers:(A) 5′-GACTGCGTACATGCAGAAC (SEQ ID NO: 5) and 5′-GATGAGTCCTGAGTAACTT,(SEQ ID NO: 6) or

b. a 151 nucleotide amplification product with the primers: (B)5′-GACTGCGTACATGCAGAAG (SEQ ID NO: 7) and 5′-GATGAGTCCTGAGTAAAAC(SEQ ID NO: 8) or

c. a 330 nucleotide amplification product with the primers: (C)5′-GACTGCGTACATGCAGACA (SEQ ID NO: 9) and 5′-GATGAGTCCTGAGTAACCA,(SEQID NO: 10)

in the genome of said plant restricted by PstI and MseI and ligated tothe oligonucleotide adapters alpha 5′-CTCGTAGACTGCGTACATGCA(SEQ ID NO: 1) CATCTGACGCATGT-5′, (SEQ ID NO: 2) and beta5′-GACGATGAGTCCTGAG (SEQ ID NO: 3) TACTCAGGACTCAT-5′. (SEQ ID NO: 4)